Insulin is a pancreatic hormone involved in the regulation of blood-glucose concentrations. For example, human, porcine, and bovine insulin, insulin analogues and mixed insulins are given to patients with insulin-dependent diabetes mellitus to control their blood-glucose concentrations.
Porcine and bovine insulin are, usually, prepared from pancreas glands. Human insulin can, semisynthetically, be prepared from porcine insulin. Alternatively, human insulin, as well as many insulin analogues, can be prepared by genetic engineering. By genetic engineering, which may, for example, be performed in bacteria or in yeast, an insulin precursor is prepared which, thereafter, is to be converted into the desired product. This conversion can be performed in different ways.
One possibility is the so-called transpeptidation where a peptide cleavage and a peptide coupling takes place consecutively in the same reaction mixture, under the same reaction conditions, vide, for example, U.S. Pat. No. 4,343,898 (Novo Industri).
Another possibility is, in the first step, to cleave the insulin precursor, vide, for example, Hoppe-Seyler's Z Physiol. Chem. 359 (1978), 799, thereafter, to isolate the intermediate product and, then, to perform the desired coupling in another reaction mixture than that used in the first step, vide, for example, Nature 280 (1979), 412.
According to EP 87,238, a transpeptidation reaction is performed in a solvent system comprising between about 75% and 97% (vol/vol) of at least one non-aqueous reaction miscible solvent including at least about 50% (vol/vol) butane-1,4-diol.
According to U.S. Pat. No. 4,579,820, the transpeptidation process is performed using an L-specific serine carboxypeptidase enzyme, for example carboxypeptidase Y.
According to U.S. Pat. No. 4,601,979 (Nordisk Insulinlaboratorium), the transpeptidation or only the peptide coupling is performed in an aqueous reaction medium substantially free of organic solvent.
According to WO 83/00504 (Nordisk Insulinlaboratorium), a porcine product was treated with carboxypeptidase A, the resulting des-alanine-B 30 insulin product was suspended in a lower alcohol, and this suspension was mixed with a solution of an L-threonine ester and trypsin. In all the specific examples, the des-alanine-B30 insulin product was isolated, either by freeze-drying or by precipitation.